title: Significance of Arg3, Arg54, and Tyr58 of L-aspartate α-decarboxylase from Corynebacterium glutamicum in the process of self-cleavage.
abstract: We have elucidated the significance of three key amino acid residues of L-aspartate α-decarboxylase that act remotely from its cleavage site for its functional self-cleavage as well as for its catalytic activity. These results provide useful fundamental information for engineering L-aspartate α-decarboxylase. L-Aspartate α-decarboxylase (ADC) from Corynebacterium glutamicum, and encoded by panD, was cloned and expressed in Escherichia coli and then purified. Three amino acid residues were found to be related to ADC self-cleavage. Mutating R3 to either A, Q, N, L, D, or E produced only the unprocessed pro-enzyme. Although mutating R54 and Y58 into A or K and A or T, respectively, partly influenced ADC self-cleavage, the specific activity of each of the four ßmutants decreased to 3.5, 4, 2.4, and 2.6 U mg(-1), respectively, compared with a specific activity of 690 U mg(-1) for the wild-type enzyme. Thus, R3 triggers ADC self-cleavage and completes the modification of the active site with assistance by R54 and Y58. These results will help to engineer ADC for improved industrial applications.
authors.affiliation: Key Laboratory of Industrial Biotechnology (Ministry of Education), School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China, wjcui@jiangnan.edu.cn.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.forename: Zengxiu
authors.initials: Z
authors.lastname: Shi
authors.forename: Yueqin
authors.initials: Y
authors.lastname: Fang
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.forename: Ning
authors.initials: N
authors.lastname: Ding
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D001120:Arginine; C019104:aspartate-alpha-decarboxylase; D005968:Glutamate Decarboxylase